The importance of carbohydrates in biologically-relevant recognition processes has only recently come to light. T. Feizi, Biochem. J. 245:1 (1987); Stults et al., Meth. Enzym. 179:167 (1989); S. Hakamori, Adv. Cancer Res. 52:257 (1989); Belvilacqua et al., Science 243:1160 (1989). These demonstrate that carbohydrates, along with proteins and nucleic acids, act as primary biologic information carriers.
The capability of carbohydrates to store and communicate information results largely from their complex stereochemistry. The multitude of stereocenters present in even small carbohydrates permits information storage analogous to that encoded in nucleic acids and in proteins. Moreover, the exposure of many carbohydrates on cell surfaces makes it possible for them to play a role in intercellular communication and recognition processes, largely on the basis of receptor-ligand interactions. Carbohydrates may be distinct macromolecules but usually are attached to other moieties such as lipids or proteins.
Examples of ligands which bind to membrane-bound receptor molecules are protein hormones and growth factors. Following binding of the cell surface receptor by the ligand, a signal is transferred to the cell interior which induces changes in the metabolism of the cell. After a certain period of time, the receptor-ligand complex is metabolically degraded and cellular metabolism returns to normal. For a review on this topic, see Alberts et al., MOLECULAR BIOLOGY OF THE CELL, 3rd edition, Garland Publishing Inc., New York, USA, page 733.
Carbohydrates on the surfaces of mammalian cells also serve as recognition domains for pathogenic agents. See, for example, J. C. Paulson, THE RECEPTORS, Vol. II, P. M. Conn (ed.), Academic Press (1985), page 131 (viruses); Stromberg et al., EMBRO J. (:2001 (1990) (bacteria); Karlsson et al., SOURCEBOOK OF BACTERIAL PROTEIN TOXINS, Alouf and Freer (ed.), Academic Press (1991), page 435 (toxins); Michel et al., Appl. Environ. Microbiol. 56:3537 (1990) (lectins). Thus, it is clear that carbohydrates play an important role in diseases which require an initial recognition of cell surface determinants.
For example, the first step in viral infection is the attachment of the virus to a host cell. This involves the specific binding viral surface proteins to receptors on the host cell membrane. These viral proteins generally are glycoproteins with a receptor binding function. Next, all or part of the viral particle is taken up into the cell and, by a complex mechanism, the viral genome is uncoated and replication proceeds.
Receptor-ligand complexes also play a part in inflammatory processes. Bradley et al., Cell 63:861 (1990). Investigations have shown, for example, that the protein ELAM 1, which is synthesized and expressed by endothelial cells in vivo, probably mediates the adhesion of leukocytes to the inflammatory focus.
The use of free oligosaccharides to diagnose and combat bacterial, viral and inflammatory disorders by competing for binding of natural ligands to the above-described receptors, has been impeded by the low affinity between receptors and the corresponding oligosaccharide ligands. Because of this low affinity, massive amounts of a competing carbohydrate are required to effectively block binding of the corresponding ligand, necessary both for diagnosis and therapy. Connolly et al., J. Biol. Chem. 257:939 (1982) (K.sub.D =.about.10.sup.-4 M for the interaction between a monovalent galactoside and the corresponding lectin).
By coupling a plurality of ligands to a "surface," an increased interaction between the receptor and ligand theoretically can be achieved. For example, the influenza virus hemagglutinin, which binds to neuraminic acid on the cell surface, has a greater affinity for its receptor when a polyvalent structure is presented. Spaltenstein et al., J. Am Chem. Soc. 113:686 (1991) (monovalent: K.sub.D =2.times.10.sup.-4 M; polyvalent: K.sub.D =3.times.10.sup.-7 M).
To date, a number of different "surfaces" have been investigated for their use in generating polyvalent structures, all of which have drawbacks: (a) liposomes- N. Yamazaki, Int. J. Biochem. 24:99 (1992); (b) polyacrylamides: (c) polylysines: and (d) sulfated polysaccharides. Each of these "surfaces" is discussed below.
Liposomes may be obtained from cell preparations or prepared from synthetic lipids. A ligand such as a carbohydrate can be held on the liposome surface by means of hydrophobic alkyl radical, attached at one point to the carbohydrate. The hydrophobic radical will be embedded in the hydrophobic membrane of the liposome, thus anchoring the complex. The disadvantages of using liposomes for this purposes are their low stability and half-life in vivo. Liposome preparations, along with other oligomeric and polymeric "surfaces" are described in WO 91/19501 and WO 91/19502.
Polyacrylamide-based polymers, prepared by polymerization of carbohydrate-containing monomers, are generally considered unsuitable as carriers of ligands for medical use. See, for example, Rathi et al., J. Polym. Sci. 29:1985 (1991) (polymerization of carbohydrate-containing acrylamide monomers for use as carrier of active substances); Nishimura et al., Macromolecules 24:4236 (1991) (copolymerized pentylglycosides with acrylamide and investigate the interaction of this macromolecule with lectins). Because the polymer portion of these compounds is composed entirely of C--C bonds, however, a crucial disadvantage arises for in vivo diagnostic and therapeutic use in that these polyacrylamide bonds are degraded in vivo to toxic metabolites and thus are not well tolerated by the subject. The relatively hydrophobic nature of these polymers also may result in unfavorable presentation of the receptor binding portion of the molecule. Moreover, both Rathi et al. and Nishimura et al. use the polymer only as carrier for a carbohydrate moiety and a pharmacologically active macromolecule.
Polylysine is composed of physiologically tolerated units. The disadvantage of polylysine, however, is in the large number of free NH.sub.2 groups which cause non-specific interactions with cells or cell surface structures. Because of the spurious interactions, it normally is impossible to achieve a site-specific effect using polylysine-containing compounds.
Conventional sulfated polysaccharides can offer only ionic interactions of low specificity. There apparently is non-specific adhesion of these compounds to any available cell surfaces and, therefore, they can offer no specific receptor blocking functions.
The interactions of dextran-fixed .beta.-blockers with .beta.-adrenoreceptors have been investigated. Pitka and Kusiak, in "Macromolecules as Drugs and as Carriers for Biologically Active Material", Tirell, Donaruma and Turek (ed.), The New York Academy of Sciences 446:249 (1985). .beta.-blockers are artificial active substances which, in this report, were linked via a spacer to the polysaccharide carrier dextran. Blocking of the .beta.-adrenoreceptors took place not by a specific receptor-ligand binding, however, but by a comparatively non-specific attachment of the blocker to the receptor.
EPA 0 089 938, EPA 0 089 939 and EPA 0 089 940 disclose sugar compounds of various chain lengths which are identical to ligands located on cell surfaces or receptors located on microorganisms. The thrust of these applications is to sue sugar compounds to bind receptors located on pathogenic microorganisms in vitro and in vivo. Thus, the diagnosis and treatment of mammalian disorders, especially those of a bacterial or viral nature are contemplated. Examples are provided for the in vitro diagnosis of bacterial infections but not for in vivo diagnosis or therapy. Also discussed are sugar compounds, coupled to a carrier either directly or via spacers, for use in preparation of antibodies, isolation of cell surface structures and disinfection of wounds. These compounds are monovalent and associate with receptors in a relatively random fashion. Thus, the binding affinities of the blockers are relatively low.
WO 92/02527 discloses macromolecules which are composed of a physiologically inert "solid carrier" and saccharide units and function as ligands for the ELAM-1 receptor. These macromolecules are suggested as useful in diagnosis of inflammatory processes in vitro but not in vivo. These compounds tend to have solubility problems, however, which limits their ability to bind the cognate receptor.
Besides toxicity and non-specific interactions, another problem with many prior art polymer "surfaces" is their relatively poor solubility. Hence, the improved binding affinity achieved by rendering carbohydrates polyvalent may be negated. Additionally, low flexibility or high steric hindrance, often associated with polymers, can decrease the finding affinity of the carbohydrate moieties.